The long-term goal is to define the molecular mechanisms that promote invasion, spread, and pathogenesis of a neurotropic herpesvirus in the mammalian nervous system. Alpha herpesviruses are remarkable because they are parasites of their natural host's peripheral nervous system. Work in this proposal focuses on how the movement of virion components inside and between neurons is regulated and how such regulation affects pathogenesis. The direction taken by the virus in a neuron after primary infection or reactivation from latency, as well the extent of spread in a neuronal circuit can be the difference between a minor peripheral infection and a devastating encephalitis. I work with pseudorabies virus (PRV), a well-studied alpha herpesvirus causing a disease of economic impact in swine, but also infecting a wide range of mammals and some birds. PRV infects both the peripheral and central nervous systems in every susceptible animal; consequently attenuated derivatives of PRV are used widely in neuroscience to map neuronal circuitry. These attenuated tracing strains carry multiple mutations that affect neuroinvasiveness. We discovered that three PRV gene products, gE, gI, and Us9, are deleted in tracing strains and dictate direction of virus spread in synaptically connected neurons. Deletion of any one of these three genes results in inability to spread from pre-synaptic to post-synaptic neuron (but not vice versa). These gene products have little affect on virus replication in the cell body. The expression of gE and gI has dramatic effects on pathogenesis (time to death and peripheral symptoms). Experiments in this proposal are directed to test hypotheses predicting how these proteins function. Aim 1: define parameters of axonal localization and delivery of PRV structural proteins by gE/gI and Us9. Aim 2: exploit multi-chamber systems to analyze and visualize virion entry, assembly and egress from neuronal cell bodies, axon shafts, and axon terminals. In Aim 3: we explore a new area of research for us, the effect of PRV infection on neuronal electrophysiology. The goal is to identify and characterize the PRV genes whose products stimulate abnormal and persistent neuronal firing of peripheral nervous system neurons.